Impact Objective• Promote biological researchwith the use of the Kanazawa
University-born revolutionary
atomic force microscopy(AFM) techniques andtechnological development
towards the next generation ofbio-AFM

Uniting
biological research
The four Heads of Division in the Bio-AFM Frontier Research Center at Kanazawa University
explain their passion for their work, the principles of the Center, and the future applications of theirresearch
PROFESSOR TAKAYUKI
UCHIHASHI, DIRECTOR OF
THE BIO-AFM FRONTIERRESEARCH CENTER
AND HEAD OF THE HIGH-SPEED
AFM DIVISIONCan you share a little about how your own
career has developed?
I helped develop atomic force microscopy(AFM) under the supervision of Professor
Seizo Morita. I learned many skills:
electronics implementation, software
programming and mechanical drawing.
During my PhD (Osaka University), I was
involved in development of non-contact
AFM (NC-AFM) working in an ultrahighvacuum. I continued research using
NC-AFM during my postdoc in the Joint
Research Center for Atom Technologyin Tsukuba. Here, I gradually shifted
my observation target from inorganic
to organic materials. When I wassubsequently working in Trinity College inDublin, I met Professor Toshio Ando ata conference in 2002. His presentation
about high-speed AFM (HS-AFM) had
a striking impact on me as I had neverimagined that AFM could achieve such
fast imaging. I then decided to change myresearch focus to biophysics using HSAFM. After joining Professor Ando’s lab at
Kanazawa University, I gained experience inthe establishment of HS-AFM and playeda direct role in some developments. I amvery proud of what we have contributed tobiological issues using HS-AFM.

What is the impact of the work thatis under way in the High-speed AFM
Division?
The mission of our Division is thedevelopment of novel instruments basedon the HS-AFM technique and using thesein the Imaging Research Division. We arealways trying to refine the present HS-AFM
technique to improve imaging speed andinvasiveness to render it a more versatile
tool. We also develop basic techniques
for functional extensions of HS-AFM. For
example, we created a wide-area imaging
technique for live-cell imaging, temperature
control systems, and interactive-mode
HS-AFM, which can manipulate moleculeswith an external force. Additionally, weare working on combining AFM withsuper-resolution optical imaging andmanipulation techniques. These are apowerful way to expand the usefulness ofHS-AFM.
PROFESSOR TOSHIO ANDO,HEAD OF THE IMAGINGDIVISION
What are the different areas of expertise ofthe staff within the Center?
We have staff members with backgrounds
in physics, applied physics, physicalchemistry, biophysics, cell biology
and bacteriology. They are skilled inelectronics design, software programming,
designing mechanical devices, control
and measurement techniques, protein

expression and purification, cell cultures,and preparation of organelles.
Have there been any key moments thathave changed your career pathway?
I gained my PhD in physics at Waseda
University and then worked as a postdoctorate researcher at the University ofCalifornia, San Francisco. After gaining atenured position in physics at Kanazawa
University I was able to set new researchtargets. I learned of the existence of AFM
from Kazuhiro Oiwa. He talked about anAFM experiment that he was planning toperform on actin-myosin interactions. I
then built an AFM instrument combinedwith a fluorescence microscope andobtained AFM images of myosin. I wasquite disappointed by the acquired images
because they were no better than electron
microscopy. I wanted to see myosin
molecules in dynamic action. I started
to develop HS-AFM in 1994. This was
initially difficult due to lack of funds but
this was solved by a large grant from the
New Energy Development Organization.
My student, Noriyuki Kodera, worked very
hard to get HS-AFM images of myosin V
and succeeded! We published the paper in
PNAS (October 2001). We started to image
myosin V interacting with actin filaments,
however the filaments broke down during
imaging as the tip-sample interaction force
was too strong. After struggling with thisproblem for three years I was able to solve
it and we finalised the development of HS-

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AFM for practical use in 2008. At last, in2010, we could watch myosin V walking onan actin filament and we published a paperin Nature.
PROFESSOR RICHARD
WONG, HEAD OF THE
MOLECULE & CELLRESEARCH DIVISION
How has your own research
career developed?
I grew up in Hong Kong. To explore theworld beyond the ‘Pearl of the Orient’, I
applied and was admitted to University ofTokyo. Initially, my dream was to become atop investment banker! It was during that
period I began to appreciate the amazing
world inside cells, with all its intrigue anduncharted complication. I was fortunate tostart working in a cell biology and anatomy
lab led by Nobutaka Hirokawa. The lab
was very energetic. People from aroundthe world worked closely together, sharing
many experimental tricks and drinking a lotof sake at parties. I enjoyed the lab workand lab life so much that I decided to makeit my career.
I moved on to work in Günter Blobel’s Labof Cell Biology at Rockefeller University.
Günter is a great mentor. He is energetic
and has a lot of motivating ideas, yet isvery open-minded and supportive of newpursuits. We discovered that vital role ofnuclear pore proteins in chromosome
segregation, spindle polarity andcentrosome homeostasis during mitosis.
I could not have made these findings
without the generous support of Günter
and Nobutaka, and the valuable help fromso many who have worked with me. I amvery fortunate.
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What kind of research is under way inyour Division?
Traffic inside a cell is as intricate as rushhour near any metropolitan area. Butdrivers know how to follow the roadways
and signs to reach their destinations.
How do diverse cellular proteins ‘read’
molecular signposts to find their way? How
does the nuclear pore gatekeeper control
the trafficking of proteins, RNAs andviruses? Our Division is divided into twogroups. Group 1 focuses on the nuclear
pore. We are observing nuclear pores incancer cells using HS-AFM. We wish toexpand HS-AFM as a diagnostic tool foruse with patients’ cells. Group 2 focuseson the bacterial organelle magnetosome,which functions as a cellular compass tonavigate along the Earth’s magnetic field.
PROFESSOR TAKESHIFUKUMA, HEAD OF THE
SUPER-RESOLUTION
AFM DIVISIONIs there any work under way in yourDivision that you would like to share?
We are working on a number of newtechniques including HS frequency
modulation AFM (FM-AFM), which werecently improved the speed from 1 min/frame to 1 sec/frame. Another is 3D
scanning force microscopy (3D-SFM)
which enables the visualisation of 3Ddistributions of hydration structuresand flexible molecular chains at a solidliquid interface. We are now working withthree different companies due to this.
Furthermore, we have also developed openloop electric potential microscopy (OLEPM). This technique is useful in observing
local distributions of electrochemical

reactions at a solid-liquid interface. We areworking with three different companies
on different subjects related to the metal
corrosion processes. Corrosion is a serious
problem in many industrial fields. The
annual economic loss caused by corrosion
amounts to 3–4 per cent of the GNP ofa country. With OL-EPM, we can directly
visualise local distribution of active
corrosion sites in real time in situ.
How has your research path developed?
I started working on AFM during mypostgraduate studies. Initially, I workedon the application of the commercial AFM
instruments to the studies on polymericmaterials. During my PhD, I studied
more advanced commercial instruments:
ultrahigh vacuum AFM for studying
organic ultrathin films at molecular-scale
resolution. As a postdoc, I started to workon improvements to the AFM instruments.
During this period, I succeeded indeveloping liquid-environment FM-AFM
that is capable of imaging atomic-scale
structures in liquid. This is my biggest
achievement so far. Most researchers
believed that FM-AFM could not work inliquid. With this success, I went abroadto Trinity College Dublin. There I mainly
worked on the development of my own
AFM system for liquid-environment
applications. I also worked on theapplications of the developed system tostudies on biological systems.

Focusing AtomicForce Microscopy
The Bio-AFM Frontier Research Center at Kanazawa University
combines cutting-edge research into developing novel techniques ofatomic force microscopy with directing these techniques towards
biological applications

Atomic force microscopy (AFM) is a
powerful technique capable of visualising
the topography of materials at an atomicresolution. At its most basic, a fine tip isused to probe the surface of the material
with detection of tip-surface interaction.
This allows the construction of a surface
image in very fine resolution not limited
by diffraction. Developments made byresearchers at Kanazawa University inJapan have refined AFM and allowed
its application to a variety of different
circumstances and, crucially, to biology.
Initially, probing a wide range of biologicalsamples was difficult as the tip involvedin AFM often destroyed the sample. Inorder to tackle such problems and focus
the technology towards end users, theBio-AFM Frontier Research Center wasestablished in 2010. Led by Professor
Takayuki Uchihashi (Director and Headof High-speed AFM), the institute has
been working hard on imaging a diverse
range of biological scenarios – from cellorganelle trafficking to individual protein
interactions and membrane interactions.
Additionally, the institute works withindustrial partners to apply their techniques
to current chemical engineering issues suchas corrosion of metal surfaces. The Centeris split into four divisions – High-speed
AFM, Super-resolution AFM, Imaging,and Molecule & Cell Research. Professor
Toshio Ando (Former Director, Head ofImaging) explains the basic aim of theCenter: ‘Our interdisciplinary centre aims
for international leadership in the biological
AFM technology and the understandingof dynamic biomolecular and cellularprocesses.’
TECHNICAL TRIUMPHS
The Bio-AFM team have been able to adapt
AFM to a variety of different applications
and thus achieve both high-resolution andreal-time images.

A major advance in this area was madeby Ando, Kodera (Member of Imaging)and Uchihashi when they created highspeed AFM (HS-AFM) with a low-invasive
imaging capability. The higher speed ofthis technique allows a more biologically
relevant look at the structural changes of amolecule. Alongside this, Professor Takeshi
Fukuma (Head of Super-resolution AFM)
developed non-contact AFM in which theprobing tip does not make direct contact
with the surface, rather relying on the effectof hydration forces to measure topography.
This prevents the degradation of the sample
by the tip, which was particularly commonfor biological samples. He is developing
several branches of AFM to image the solidliquid interface.
GROUNDBREAKING BIOLOGYThe Bio-AFM Frontier Research Center
was primarily founded in order to further
the application of these AFM-derived
techniques to the life sciences. To thisend, Professor Richard Wong is leading
the Molecule & Cell Research Division thatis investigating two key imaging areas.
First is the focus on the nuclear pore anddetermine how it selects the molecules andproteins that pass through it. Secondly they
are investigating the bacterial organelle
known as the magnetosome which allows
certain bacteria to navigate by the Earth’s
magnetic field. This organelle contains
magnetite crystals surrounded by a lipid
bilayer and is thus an ideal structure forinvestigation through AFM means.
Together, the four Divisions of Bio-AFM
are working to develop and hone toolsthat promise to greatly expand our ability
to investigate fine molecular structures.
From visualising fundamental atomistic
phenomena to uncovering the complexityof protein functions, Bio-AFM will generate
new levels of understanding of molecularand atomic interactions.

Project Insights
FUNDING
KAKENHI Basic Research (S)#24227005 (PI, Toshio Ando) – New
structural biology to be opened byhigh-speed atomic force microscopy(2012 – 2017) • JST/CREST (PI, ToshioAndo) – Dynamic structural biology ofprotein machineries driven by ATP/GTP(2013 – 2019) • KAKENHI Research on
Innovative Area #26119003 (PI, ToshioAndo) – Development of advanced highspeed AFM and analysis of proteins’
action mechanism (2014 – 2019)AIMS
Develop technology for atomic/
molecular-level analysis • Pioneer
biological phenomena and biologicalsamples for bio-AFM studies • Develop
instruments for elucidating biologicalphenomena • Elucidate biologicalphenomena at the single-molecule level
CONTACTToshio Ando
Head of Imaging Division at theBio-AFM Frontier Research Center,
Kanazawa University
T: +81 76 2645663
E: tando@staff.kanazawa-u.ac.jp
W: http://www.se.kanazawa-u.ac.jp/
bioafm_center/index.htm
BIO
Professor Toshio Ando received a Dc.
Sci. degree in Physics from WasedaUniversity, Japan, in 1980. He wasa postdoctoral fellow at Universityof California, San Francisco, before
becoming an Assistant Professor at thesame institution. He returned to Japan
in 1986 to work at Kanazawa University
where his research focuses on highspeed AFM and its application toproteins and cells. Ando is the founderof the Bio-AFM Frontier ResearchCenter.

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